Purpose: :
Melanopsin is a novel retinal G–protein coupledreceptor (GPCR) which is expressed in a subset of intrinsicallyphotosensitive retinal ganglion cells (ipRGCs) in the innerretina of mammals. The light–dependent response in ipRGCshas been demonstrated to be most sensitive to 480nm light, andmelanopsin is also necessary for this response. In in vitro systems melanopsin can be expressed and reconstitutedwith 11–cis–retinal to form a functional photopigmentthat is able to activate a heterotrimeric G–protein ina light–dependent manner. These properties of melanopsinsuggests that it may be the primary photopigment initiatingthe light response in ipRGCs, but unlike other vertebrate visualpigments, melanopsin’s absorbance maximum of 420nm whensolubilized in detergent is drastically blue–shifted fromthe wavelength sensitivity of ipRGCs. Although this type ofspectral difference between in vivo and in vitro data has notbeen observed in vertebrate visual pigments, it has been reportedin some invertebrate visual pigments. In this investigationwe demonstrate that melanopsin solubilized in detergent is sensitiveto the ionic strength of the solution.

Methods: :
The expressed melanopsin has a 1D4 epitope tag (thelast 15 amino acids of Bovine Rhodopsin) appended to its C–terminus.Expressed melanopsin is reconstituted with chromophore and thenpurified using immuno–affinity column chromatography.Purified melanopsin is analyzed using a spectrophotometer todetermine the wavelength absorbance spectrum for the photopigment.Purified membranes containing melanopsin are used in a filter–bindingassay to determine the photopigments rate of G–proteinactivation

Results: :
Solubilized in a detergent solution with high or lowNaCl concentration, melanopsin forms two spectrally distinctisoforms of the photopigment, with absorbance maxima of 420nmand 480nm respectively. We also show that heterologously expressedmelanopin in the plasma membrane of COS cells activates Gt withan increased sensitivity to 480nm over 420nm.

Conclusions: :
These data show that the heterologously expressedmelanopsin can form a photopigment that matches the in vivowavelength sensitivity, and further suggests that melanopsinis the photopigment that initiates the photoresponse of ipRGCs.These results also suggest that along with increased amino acidsequence similarity to invertebrate opsins, melanopsin may alsoshare some biochemical similarities.